Milk clotting enzymes are widely used in the cheese industry to provide a curd of the major milk proteins. Commercially available milk clotting enzymes include native enzymes derived from microbial or animal tissue sources, or the enzymes may be provided as gene products of recombinant cells expressing a milk clotting enzyme of animal or microbial origin.
Native milk clotting enzymes of animal origin are isolated by extraction from animal tissues containing one or several of these enzymes. Thus, animal milk clotting enzymes include several enzymes of the group of aspartic endopeptidases having molecular weights which are in the range of about 35,000 to 42,000 daltons (group 3.4.23 according to the Enzyme Nomenclature, 1992 of the International Union of Biochemistry and Molecular Biology, IUBMB) such as pepsin A (3.4.23.1) and gastricsin (3.4.23.3) which is excreted into the gastric juice of vertebrates including ruminants, pigs and humans, and chymosin (3.4.23.4) which is a predominantly neonatal gastric enzyme with high milk clotting activity, excreted in mammals. The molecular weights of animal milk clotting enzymes are in the range of about 35,000 to about 42,000 daltons. Thus, chymosin has a calculated molecular weight of 35,652 daltons.
The primary industrial source of native animal milk clotting enzymes are stomachs of calves and adult cattle in which essentially all of the in-vivo milk clotting activity is associated with the presence in the gastric juice of chymosin and pepsin A. However, when produced in the stomach tissue cells, these enzymes occur as enzymatically inactive pre-enzymes which are designated pre-prochymosin and pre-pepsinogen A, respectively. When chymosin is excreted, an N-terminal fragment is cleaved off to give prochymosin including a pro-fragment. Prochymosin is an essentially inactive form of the enzyme which, however, under acidic conditions becomes activated to the active chymosin molecule by removal of the pro-fragment. This activation takes place in-vivo in the gastric lumen under appropriate pH conditions. Pepsinogen A is activated into the active enzyme by partial hydrolysis under acidic conditions.
Pseudochymosin is the designation of a chymosin species where only part of the pro-fragment (amino acid residues 1-27) is removed. Pseudochymosin will e.g. occur in an extract which has been exposed to a low pH, such as a pH of 2. Pseudochymosin has enzyme activity and is stable at low pH but is processed to chymosin at higher pH. The isoelectric point of pseudochymosin is not known, but it is assumed to be higher than 4.9.
Recently, animal chymosin that is produced in recombinant microorganisms including filamentous fungi has been introduced into the industrial market. Such recombinantly manufactured milk clotting enzyme products are also referred to as fermentation produced chymosin or rennet.
In addition to the above milk clotting enzymes of animal origin several natively produced microbial enzymes are used in the dairy industry. Such enzymes are referred to as microbial milk clotting enzymes or microbial coagulants in the following. Examples of such enzymes include Rhizomucor (previously Mucor) miehei proteases including destabilized, i.e. oxidized Rhizomucor miehei protease, Mucor pusillus protease and Endothia parasitica protease.
Preparations or compositions containing native milk clotting enzymes of animal origin are prepared industrially by extraction from stomach tissues, in particular from ruminants including calves and adult cattle. Enzyme-containing crude extracts contain chymosin species including precursors, and pepsin species in ratios which depend primarily on the age of the animal. Thus, the distribution between chymosin and pepsin in stomachs from young calves is typically about 80:20 to 90:10 whereas in stomachs from adult cattle it is typically about 25:75. It will be understood that intermediate distributions between these enzyme species may be found in older calves and young cattle. As an example, the above ratio in extracts from these animals is typically in the order of 50:50.
Conventionally, commercial products containing milk clotting enzymes of animal origin is manufactured by a multistep, time consuming process which typically include the following steps: (i) preparing a crude enzyme-containing extract by extracting comminuted, frozen or dried calf or cattle stomachs with water, (ii) transforming the proenzymes into the active enzymes, (iii) a clarification step wherein a flocculant is added to facilitate the subsequent filtration step, (iv) concentration steps, (v) repeated clarification, (vi) further filtration step to remove precipitated impurities, (vii) adjusting salt and preservative concentration, (viii) adjusting the enzymatic strength and composition to obtain the finished product which is usually referred to as rennet. Prior to packaging, the rennet product may be subjected to a final filtration step including a sterile filtration. This conventional process of manufacturing rennet involves a high consumption of chemical agents and energy.
The entire conventional process of rennet manufacturing may take about one week. It will be understood that such conventionally manufactured enzyme preparations will contain a mixture of chymosin and pepsin, the latter enzyme being much less active than chymosin in respect to milk clotting.
However, chymosin is considered in the dairy industry as far the best milk clotting enzyme for manufacturing of cheese with respect to specific clotting activity, curd formation, cheese texture and flavour and yield of cheese. The distribution of milk clotting enzymes, i.e. the composition of a given batch of animal rennet may vary considerably depending i.a. on the animal stomach raw material. If a conventional rennet having a relatively high proportion of chymosin is desired, stomachs from young calves is the preferred raw material. Such products may be designated calf rennet. Rennet products having lower proportions of chymosin may be manufactured on the basis of stomachs from older calves or from adult cattle (ox rennet). Naturally, rennet products having intermediate enzyme chymosin content may be obtained e.g. by mixing a calf rennet and an ox rennet. The strength of a crude extract as described above is typically in the range of 5 to 30 Chr. Hansen units (CHU)/mL as defined below. Typically conventionally manufactured commercial liquid rennet products has an enzymatic strength in the range of 40 to 100 CHUs/mL.
The above conventional rennet manufacturing process has several essential drawbacks. Firstly, the process is time consuming and labour-intensive; secondly, the provision of rennet having high contents of chymosin requires use of stomachs from young calves which is a relatively scarce and costly raw material; thirdly, the process requires use of clarifying agents which add to the production costs; fourthly part of the process takes place at pH values where microbial growth is not inhibited, which may cause spoilage problems and furthermore, part of the process is at pH values where the chymosin has a reduced stability; fifthly, the process results in large quantities of contaminated waste-water and sixthly, the resulting rennet may be less suitable as the basis for production of powdered rennet products with a very high milk clotting activity due to its relatively low enzymatic activity (strength) and finally, it is a significant drawback that the liquid rennet products have low strengths which implies significant transportation costs.
In the prior art some of the above problems have been addressed e.g. by suggesting processes whereby chymosin and pepsin are separated. Thus, WO 88/02220 discloses a method for separating chymosin from a liquid containing chymosin and pepsin, comprising adjusting the enzyme-containing liquid to a pH of about 3.8 to about 5.2, preferably in the range of 4.4 to 4.5, and to a conductivity of about 2 to about 19 mS/cm, and contacting this liquid with an equilibrated anion exchange medium to bind pepsin, recovering the chymosin in the liquid resulting after contact with the exchange medium and removing the pepsin from the exchange medium. However, this process results in a fraction where chymosin is present in an amount which is generally lower than that of the starting enzyme-containing liquid and the process is based on anion exchange at a pH where chymosin is unstable and which allows microbial growth. Furthermore, the anion exchange medium as disclosed is a weak DEAE-cellulose ion exchanger the performance of which under the disclosed conditions will be affected by small variations in the pH or conductivity of the extract applied to the weak exchanger.
In WO 90/15866 is disclosed a method of recovering chymosin from an aqueous solution which additionally contains pepsin, comprising adding to the aqueous solution, polyethylene glycol (PEG) and an inorganic salt so as to form a two-phase system which after separation into a chymosin and pepsin-rich PEG phase and an enzyme-poor salt phase, and contacting the PEG phase with an ion exchange resin under conditions where the chymosin is bound to the resin, and recovering the chymosin from the resin. Although this process may result in a rennet product essentially only containing chymosin, it involves several technological drawbacks which contribute significantly to the production costs: (i) the use of PEG during the process requires strict measures be taken to remove the PEG, (ii) PEG and inorganic salt in the high amounts to be used are costly, (iii) in order to dissolve the added salt, it is required to use energy to warm the solution, (iv) the two phases must be separated by centrifugation (v) after the ion exchange step, the PEG must either be recycled or discharged, and (vi) according to that disclosure it is necessary to regenerate the resin after each batch.
The present invention provides a novel one-phase ion exchange-based process of separating chymosin and pepsin from a crude extract of animal tissues containing these milk clotting enzymes that is surprisingly efficient with regard to separating and concentrating the enzymes and to obtaining a high yield of enzymes, and which in comparison to the prior art methods is simple and cost-effective.
However, it has been found that rennet products containing milk clotting enzymes of animal origin that are provided by an ion exchange process may be prone to reduction in their enzymatic activity, assumingly due to oxidation of certain amino acid residues. This phenomenon of oxidative enzyme destabilization is known in the art in connection with microbial coagulants, and is in fact utilized in the manufacturing of so-called destabilized milk clotting proteases of microbial origin, which is oxidized to a level where they are readily inactivated during the pasteurization of the whey. It is also known to add stabilizing agents to such proteases in order to preserve their activity up to their use. Thus, DE 3234761 discloses solutions of such fungal proteases stabilized by the addition of at least 0.1 wt % methionine.
Milk clotting enzymes may e.g. be exposed to oxidizing agents such as e.g chlorine or cleaning agent residues present in the water used during the manufacturing of rennet products or in the dairies. In contrast to the rennet products provided herein, conventionally manufactured animal milk clotting enzyme extracts (rennets) are not very sensitive to oxidation due to their high content of impurities which are readily oxidized.
It has now surprisingly been found that it is possible to protect animal milk clotting enzymes having a low content of impurities by the addition of compounds which are readily oxidized, including methionine. Therefore, it is an important aspect of the present invention to provide liquid and powdered rennet products comprising a milk clotting enzyme and a stabilizing compound which improve the stability of the enzymes against oxidative inactivation. In the present context, rennet products having a low content of impurities include rennets manufactured on the basis of enzyme-containing animal tissues, e.g. in accordance with the process of this invention or by an other process leading to a rennet product with a low content of impurities which are readily oxidizable, and milk clotting enzymes expressed by a recombinant microorganism (fermentation product rennets).